Matsuda K, Araki E, Yoshimura R, Tsuruzoe K, Furukawa N, Kaneko K, Motoshima H, Yoshizato K, Kishikawa H, Shichiri M
Department of Metabolic Medicine, Kumamoto University School of Medicine, Japan.
Diabetes. 1997 Mar;46(3):354-62. doi: 10.2337/diab.46.3.354.
Insulin receptor substrate 1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream. We have previously shown that the levels of IRS-1 mRNA varied in different tissues. To elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and transacting factors in CHO and HepG2 cells. Using the chloramphenicol acetyltransferase (CAT) assay with the various deletion mutants of the IRS-1 promoter-CAT fusion plasmids, several regions responsible for positive or negative regulation in each cell line were identified. A region from -1645 to -1585 bp, which regulated expression negatively in CHO cells and positively in HepG2 cells, was further analyzed. Within this region a fragment from -1645 to -1605 bp upregulated the IRS-1 promoter only in HepG2 cells, whereas a fragment from -1605 to -1585 bp downregulated only in CHO cells. In the gel mobility shift assay, several nuclear proteins that bind to these fragments were detected, and among them, two nuclear proteins that bind to a potential E box (nucleotide [nt] -1635 to -1630) and two nuclear proteins that bind to a potential C/EBP binding site (nt -1599 to -1591) were identified in HepG2 and CHO cells, respectively. CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell-specific regulation of the IRS-1 gene. We therefore concluded that the two nuclear proteins that bind to the E box regulate IRS-1 gene expression positively in HepG2 cells and the two nuclear proteins that bind to the C/EBP binding site regulate it negatively in CHO cells.
胰岛素受体底物1（IRS-1）是胰岛素受体酪氨酸激酶的主要底物之一，介导下游多种胰岛素信号。我们之前已经表明，IRS-1 mRNA的水平在不同组织中有所不同。为了阐明IRS-1组织特异性调控的分子机制，我们研究了CHO和HepG2细胞中的顺式作用元件和反式作用因子。使用氯霉素乙酰转移酶（CAT）分析以及IRS-1启动子-CAT融合质粒的各种缺失突变体，确定了每个细胞系中负责正调控或负调控的几个区域。对一个从-1645至-1585 bp的区域进行了进一步分析，该区域在CHO细胞中负调控表达，而在HepG2细胞中则正调控表达。在该区域内，一个从-1645至-1605 bp的片段仅在HepG2细胞中上调IRS-1启动子，而一个从-1605至-1585 bp的片段仅在CHO细胞中下调。在凝胶迁移率变动分析中，检测到几种与这些片段结合的核蛋白，其中，在HepG2细胞和CHO细胞中分别鉴定出两种与潜在E盒（核苷酸[nt] -1635至-1630）结合的核蛋白以及两种与潜在C/EBP结合位点（nt -1599至-1591）结合的核蛋白。使用在E盒或C/EBP结合位点处突变的启动子进行的CAT分析表明，这些序列负责IRS-1基因的细胞特异性调控。因此，我们得出结论，与E盒结合的两种核蛋白在HepG2细胞中正向调节IRS-1基因表达，而与C/EBP结合位点结合的两种核蛋白在CHO细胞中负向调节该基因表达。
